Bottom-up proteomics
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Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and
post-translational modification Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
s by proteolytic digestion of proteins prior to analysis by
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
. The major alternative workflow used in
proteomics Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
is called
top-down proteomics Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods ...
where intact proteins are purified prior to digestion and/or fragmentation either within the mass spectrometer or by 2D electrophoresis. Essentially, bottom-up proteomics is a relatively simple and reliable means of determining the protein make-up of a given sample of cells, tissues, etc. In bottom-up proteomics, the crude protein extract is enzymatically digested, followed by one or more dimensions of separation of the peptides by
liquid chromatography In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
coupled to mass spectrometry, a technique known as
shotgun proteomics Shotgun proteomics refers to the use of bottom-up proteomics techniques in identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun seq ...
. By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database or annotated peptide spectral in a peptide spectral library, peptides can be identified and multiple peptide identifications assembled into a protein identification.


Advantages

For high throughput bottom-up methods, there is better front-end separation of peptides compared with proteins and higher sensitivity than the (non-gel) top-down methods.


Disadvantages

There is limited protein sequence coverage by identified peptides, loss of labile PTMs, and ambiguity of the origin for redundant peptide sequences. Recently the combination of bottom-up and top-down proteomics, so called middle-down proteomics, is receiving a lot of attention as this approach not only can be applied to the analysis of large protein fragments but also avoids redundant peptide sequences.


See also

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Protein mass spectrometry Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instru ...
*
Shotgun proteomics Shotgun proteomics refers to the use of bottom-up proteomics techniques in identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun seq ...
*
Top-down proteomics Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods ...


References

Mass spectrometry Proteomics {{bioinformatics-stub